Cancer Driver Research Articles

Abstract LB182: Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520

Abstract Introduction. ROS1 fusions are recurrent oncogenic drivers in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) crizotinib, entrectinib, and repotrectinib have been approved for the treatment of ROS1-positive NSCLC. Response to these TKIs can be limited by emergence of drug resistance, with ~40% of patients acquiring on-target ROS1 mutations at progression on crizotinib. NVL-520 is an investigational, brain-penetrant, and TRK-sparing ROS1 TKI designed to maintain activity for ROS1 mutations that confer resistance to approved therapies. To investigate suppression of resistance by NVL-520, we performed two N-ethyl-N-nitrosourea (ENU) mutagenesis screens and determined the frequency and identity of resistance mutations following treatment with NVL-520 versus earlier-generation ROS1 TKIs. Methods. Cells were treated with ENU for 1 day followed by crizotinib, entrectinib, repotrectinib, or NVL-520 for 28 days. Frequency of resistance indicates the fraction of wells that proliferated on day 28 despite continuous TKI treatment. Resistance mutations in the ROS1 kinase domain were identified by Sanger sequencing. Resistant clones harboring ROS1 kinase-domain mutations were expanded and used to profile the inhibitory activity of ROS1 TKIs. Results. The first ENU screen was performed on Ba/F3 CD74-ROS1, simulating a first-line treatment setting. NVL-520 at an unbound drug concentration (Cunbound) of 8.2 nM yielded 1 resistant clone among 480 replicate wells (frequency of resistance = 0.2%). The second screen was performed on Ba/F3 CD74-ROS1 G2032R, which is the most common point mutation after progression on previous-generation therapies, simulating a later-line treatment setting. NVL-520 at Cunbound of 41 nM yielded 6 resistant clones among 480 replicate wells (frequency of resistance = 1.3%). These NVL-520 concentrations (8.2 - 41 nM) were well within its average Cunbound of 166 nM at the recommended phase 2 dose (RP2D) of 100 mg once daily. None of the 7 resistant clones harbored ROS1 kinase-domain mutations, suggesting an off-target resistance mechanism. Crizotinib, entrectinib, and repotrectinib at their respective average human Cunbound yielded frequencies of resistance ranging from >33% to 100% in at least one of the screens, indicating that they were less effective than NVL-520 at suppressing resistance in these screens. Conclusion. NVL-520 was highly effective at suppressing resistance in ENU mutagenesis screens simulating first-line (CD74-ROS1) and later-line (CD74-ROS1 G2032R) treatment. The screens did not reveal any novel ROS1 mutations that could confer resistance to NVL-520 at the RP2D. The broad preclinical activity against ROS1 and diverse ROS1 mutations, together with its brain penetrance and TRK-sparing design, suggests NVL-520 as a potential best-in-class ROS1 therapy. Citation Format: Anupong Tangpeerachaikul, Franklin Gu, Henry E. Pelish. Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB182.

Abstract LB217: Novel organoid models of lineage plasticity in castration resistant prostate cancer

Abstract The development of next-generation AR-targeted drugs, such as abiraterone and enzalutamide, has improved survival of patients with castration-resistant prostate cancer (CRPC). However, many patients eventually progress to therapy-resistant lethal prostate cancer. CRPC exhibits variant cellular states, including AR-pathway active prostate cancer (ARPC), AR activity-low prostate cancer (ARLPC), neuroendocrine prostate cancer (NEPC), amphicrine prostate cancer co-expressing AR and neuroendocrine genes (AMPC), and double-negative prostate cancer (DNPC) lacking both AR expression and neuroendocrine differentiation, indicating that lineage plasticity facilitates therapeutic resistance. While molecular characteristics of ARPC and NEPC are well described, other common forms of CRPC, such as DNPC which occurs in 20-25% of CRPC patients, remain poorly understood. Therefore, characterization of pre-clinical models of DNPC and identification of novel regulators of lineage plasticity in DNPC are urgent necessities for the development of effective treatment for lethal prostate cancer.In our studies, we have generated a new mouse model of CRPC in which Pten and Smad4 are deleted in distal luminal cells upon tamoxifen administration (Nkx3.1CreERT2/+; Ptenfl/fl; Smad4fl/fl; R26R-eYFP/+, NPS). We observed a molecular transition from ARPC to ARLPC and DNPC in the prostate tumors derived from NPS mice during tumor progression. Moreover, a combination of castration and enzalutamide treatment promoted a partial epithelial-mesenchymal transition (EMT) in DNPC. To identify the evolutionary trajectories of cancer progression and drivers of lineage plasticity in CRPC, we have established organoid lines from the prostate tumor cells of NPS mice. Single-cell RNA-sequencing (scRNA-seq) and marker validation analyses shown that these organoid lines display overt lineage plasticity, corresponding to ARPC, ARLPC, partial EMT-absent DNPC, and partial EMT-present DNPC. Orthotopic grafts derived from the established NPS organoid lines show consistent molecular phenotypes and confirm that AR-targeted therapies facilitate partial EMT in vivo. Furthermore, cross-species bioinformatic analyses using scRNA-seq datasets indicate that the established NPS organoid lines partially recapitulate the spectrum of lineage plasticity in human CRPC.In summary, we have established prostate cancer organoid lines from a novel mouse model of CRPC, which not only provide large and reproducible sources of two distinct forms of DNPC (partial EMT-absent DNPC and partial EMT-present DNPC) for molecular analyses, but also can capture the regulatory complexity of lineage plasticity in human CRPC. Therefore, NPS mice provide an excellent pre-clinical model of CRPC, especially DNPC, to identify novel regulators that drive lineage plasticity and to pursue assays for development of candidate drugs that can inhibit or even possibly convert therapy-resistant forms of CRPC to therapy-sensitive ones. Citation Format: Jinqiu Lu, Michael M. Shen. Novel organoid models of lineage plasticity in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB217.

Abstract LB007: A systematic evaluation of the therapeutic potential of Endogenous-ADAR base editors in cancer prevention and treatment

Abstract Base editing encompasses techniques that efficiently alter specific nucleotides at the DNA or RNA level. Initially explored for inherited diseases, these techniques hold promise for addressing various genetically driven disorders caused by single nucleotide variants (SNVs). The precise programmability of base editors (BEs) for specific sequences allows customization for rare genetic variants, tailoring them to individual patients within affordability and delivery constraints. Cancer stems from the accumulation of mutations. However, the relevance of BEs in cancer therapy is doubted due to the limited types of mutations they can address within tumors. Yet, their untapped potential in the realm of cancer treatment invites exploration. BEs utilize a modified form of a deaminase enzyme to catalyze the conversion of one nucleotide to another by removing an amino group. A 'classic' BE consists of a deaminase, a Cas nuclease, and a guide RNA (gRNA) ensuring target specificity through Watson-Crick base pairing. An alternative RNA BE, known as Endogenous-ADAR, involves designing a gRNA to recruit native Adenosine Deaminases Acting on RNA (ADAR), responsible for extensive A-to-I(G) editing in mammals. Due to its reliance on ADAR capabilities, Endogenous-ADAR exclusively targets G>A SNVs at the RNA level. Notably, RNA editing, occurring before splicing, provides the flexibility to target all gene regions. Also, editing the RNA sequence is considered 'safer' in the event of an off-target error. Our objective is to systematically explore the potential of Endogenous-ADAR for cancer prevention and treatment. Our first approach evaluates Endogenous-ADAR's potential to revert germline mutations in cancer predisposition genes (CPGs) for cancer prevention. Focusing on CPGs recommended for pediatric genetic testing by NCBI, associated with cancer predisposition disorders, our findings indicate that among the 2,820 SNVs examined, 566 (20%) are suitable for Endogenous-ADAR. Remarkably, 88% of correctable variants show no off-target sites, indicating safe therapeutic targets. Further examining pathogenic high-penetrance germline variants in adults, 48 (20%) of the 239 SNVs examined are correctable using Endogenous-ADAR. Second, we estimate the potential to correct cancer driver mutations. Examining 5,913 driver mutations from 2,010 patients representing 36 cancer types, which underwent whole genome sequencing (WGS) in the PCWAG project, we find that 955 (16%) are suitable for Endogenous-ADAR. In 729 (36%) of patients, at least one driver mutation is correctable, and in 91 (5%), all known driver mutations are correctable. Aligned with the swift incorporation of WGS in oncology, our systematic exploration portrays a favorable landscape of correctable mutations in cancer. This points to the future possibility of leveraging the unique capabilities of base editing, particularly Endogenous-ADAR, for clinical cancer risk/prevention and treatment outcomes. Citation Format: Rona Merdler-Rabinowicz, Ariel Dadush, Sumeet Patiyal, Padma Sheila Rajagopal, Gulzar Daya, Alejandro Schäffer, Eli Eisenberg, Eytan Ruppin, Erez Y. Levanon. A systematic evaluation of the therapeutic potential of Endogenous-ADAR base editors in cancer prevention and treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB007.

Abstract SY07-02: Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut

Abstract The epithelial monolayer of the intestine is one of the fastest renewing tissues of the human body. Every week, the entire epithelium is replaced by intestinal stem cells (ISCs) that reside in crypt-like structures within the monolayer. Within these crypts, a small number of ISCs continuously divide to provide all specialized cell types necessary to maintain normal intestinal homeostasis, while simultaneously competing with each other for a position within the crypt bottom. This competition, characterized by neutral loss-and-replacement events, can be drastically disturbed when one of the ISCs acquires a mutation that confers a competitive advantage. As a result, mutant ISCs will replace all normal ISCs, colonize the intestinal crypt, and facilitate the initiation of colorectal cancer (CRC). CRC development is a stepwise process characterized by the accumulation of mutations in cancer driver genes over time. Almost 80% of all CRCs present with early mutations in the tumor suppressor gene APC, leading to constitutive activation of the Wnt pathway and the formation of pre-malignant adenomas. We have previously demonstrated that, within individual crypts, Apc-mutant ISCs have a competitive advantage over normal ISCs, although the mechanism underlying this unequal competition remained unclear. Recently, we revealed that Apc-mutant ISCs in fact act as "supercompetitors" by secreting Wnt antagonists such as NOTUM, WIF1 and DKK2, that actively disadvantage neighboring wild type ISCs by forcing them do differentiate. We demonstrated that boosting wild type fitness by downstream pharmacological activation of the Wnt signaling pathway by GSK3β-inhibitor lithium rendered wild type ISCs resistant to the Wnt antagonists, reduced the number of Apc-mutant crypts, and prevented adenoma formation. Together, this work highlights that modulating cell competition between normal and mutant cells within the crypt can provide a novel approach to prevent CRC development. Our findings are particularly relevant for familial adenomatous polyposis (FAP) patients, that carry germline mutations in APC and are predisposed to the development of CRC. Based on our fundamental discoveries, we are currently performing a clinical trial to assess the chemopreventive effect of lithium on the expansion of APC-mutant clones in FAP patients [ClinicalTrials.gov: NCT05402891]. Importantly, it is becoming increasingly evident that intestinal adenomas do not form from a single Apc-mutant crypt, but require fields of multiple mutant crypts to facilitate tumor initiation. Although field cancerization by enhanced crypt fission has been observed for several oncogenic mutants in the gut (e.g. KrasG12D, PIK3CAH1047R), Apc-mutant cells are often contained within individual crypts. This indicates that multiple independent mutant clones may act cooperatively to facilitate tumorigenesis, thereby pointing towards a polyclonal origin of intestinal adenomas. Our current work investigates how Apc-mutant clones interact by modifying their local environment through reciprocal signalling with stromal and epithelial cells to escape the anatomical constraints of the crypt, thereby unlocking their oncogenic potential in the gut. Ultimately, unravelling the molecular mechanisms underlying clonal cooperation will inform potential chemoprevention strategies for CRC. Citation Format: Sanne van Neerven. Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY07-02.

LTK mutations responsible for resistance to lorlatinib in non-small cell lung cancer harboring CLIP1-LTK fusion

The CLIP1-LTK fusion was recently discovered as a novel oncogenic driver in non-small cell lung cancer (NSCLC). Lorlatinib, a third-generation ALK inhibitor, exhibited a dramatic clinical response in a NSCLC patient harboring CLIP1-LTK fusion. However, it is expected that acquired resistance will inevitably develop, particularly by LTK mutations, as observed in NSCLC induced by oncogenic tyrosine kinases treated with corresponding tyrosine kinase inhibitors (TKIs). In this study, we evaluate eight LTK mutations corresponding to ALK mutations that lead to on-target resistance to lorlatinib. All LTK mutations show resistance to lorlatinib with the L650F mutation being the highest. In vitro and in vivo analyses demonstrate that gilteritinib can overcome the L650F-mediated resistance to lorlatinib. In silico analysis suggests that introduction of the L650F mutation may attenuate lorlatinib-LTK binding. Our study provides preclinical evaluations of potential on-target resistance mutations to lorlatinib, and a novel strategy to overcome the resistance.

Early-Stage Non-Small Cell Lung Cancer: Prevalence of Actionable Alterations in a Monocentric Consecutive Cohort.

Early-stage (ES) non-small cell lung cancer (NSCLC) is diagnosed in about 30% of cases. The preferred treatment is surgery, but a significant proportion of patients experience recurrence. Neoadjuvant and adjuvant chemotherapy has a limited clinical benefit. EGFR tyrosine kinase inhibitors and immunotherapy have recently opened new therapeutic scenarios. However, only a few data are available about the ES-NSCLC molecular landscape and the impact of oncogene addiction on therapy definition. Here, we determined the prevalence of the main lung cancer driver alterations in a monocentric consecutive cohort. Molecular analysis was performed on 1122 cases, including 368 ES and 754 advanced NSCLC. The prevalence of actionable alterations was similar between early and advanced stages. ES-NSCLC was significantly enriched for MET exon-14 skipping alterations and presented a lower prevalence of BRAF p.(V600E) mutation. PD-L1 expression levels, evaluated according to actionable alterations, were higher in advanced than early tumors harboring EGFR, KRAS, MET alterations and gene fusions. Taken together, these results confirm the value of biomarker testing in ES-NSCLC. Although approved targeted therapies for ES-NSCLC are still limited, the identification of actionable alterations could improve patients' selection for immunotherapy, favoring the enrollment in clinical trials and allowing a faster treatment start at disease recurrence.

Minichromosome Maintenance Complex Component 5 Modified by Mettl3 Inhibits the Proliferation of Liver Cancer by Regulating PI3K/AKT/mTOR Axis

Hepatocellular carcinoma (HCC) is a major contributor to global cancer-related deaths. The chromatin binding protein MCM5, part of the MCM family, plays a crucial role in regulating DNA replication, a key driver of cancer. Database analysis revealed elevated MCM5 levels in HCC, associated with shorter patient survival. Silencing MCM5 impedes liver cancer cell proliferation by halting the cell cycle at G1 phase. In vivo experiments confirm this effect, demonstrating that MCM5 knockdown suppresses HCC growth. Mechanistic studies unveil MCM5′s impact on HCC development via the PI3K/AKT/mTOR signaling pathway. Reversing liver cancer growth is possible by adding AKT agonist SC79. Additionally, inhibiting mettl3 with stm2457 downregulates MCM5, further suppressing liver cancer growth. In summary, high MCM5 expression in liver cancer correlates with poor prognosis and drives disease progression. Targeting MCM5 with mettl3 inhibitors presents a promising therapeutic strategy for HCC.

The MET Oncogene: Thirty Years of Insights into Molecular Mechanisms Driving Malignancy.

The discovery and subsequent research on the MET oncogene's role in cancer onset and progression have illuminated crucial insights into the molecular mechanisms driving malignancy. The identification of MET as the hepatocyte growth factor (HGF) receptor has paved the path for characterizing the MET tyrosine kinase activation mechanism and its downstream signaling cascade. Over the past thirty years, research has established the importance of HGF/MET signaling in normal cellular processes, such as cell dissociation, migration, proliferation, and cell survival. Notably, genetic alterations that lead to the continuous activation of MET, known as constitutive activation, have been identified as oncogenic drivers in various cancers. The genetic lesions affecting MET, such as exon skipping, gene amplification, and gene rearrangements, provide valuable targets for therapeutic intervention. Moreover, the implications of MET as a resistance mechanism to targeted therapies emphasize the need for combination treatments that include MET inhibitors. The intriguing "flare effect" phenomenon, wherein MET inhibition can lead to post-treatment increases in cancer cell proliferation, underscores the dynamic nature of cancer therapeutics. In human tumors, increased protein expression often occurs without gene amplification. Various mechanisms may cause an overexpression: transcriptional upregulation induced by other oncogenes; environmental factors (such as hypoxia or radiation); or substances produced by the reactive stroma, such as inflammatory cytokines, pro-angiogenic factors, and even HGF itself. In conclusion, the journey to understanding MET's involvement in cancer onset and progression over the past three decades has not only deepened our knowledge, but has also paved the way for innovative therapeutic strategies. Selective pharmacological inactivation of MET stands as a promising avenue for achieving cancer remission, particularly in cases where MET alterations are the primary drivers of malignancy.

Characterization of tumor evolution by functional clonality and phylogenetics in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a molecularly heterogeneous solid malignancy, and its fitness may be shaped by how its tumor cells evolve. However, ability to monitor tumor cell evolution is hampered by the presence of numerous passenger mutations that do not provide any biological consequences. Here we develop a strategy to determine the tumor clonality of three independent HCC cohorts of 524 patients with diverse etiologies and race/ethnicity by utilizing somatic mutations in cancer driver genes. We identify two main types of tumor evolution, i.e., linear, and non-linear models where non-linear type could be further divided into classes, which we call shallow branching and deep branching. We find that linear evolving HCC is less aggressive than other types. GTF2IRD2B mutations are enriched in HCC with linear evolution, while TP53 mutations are the most frequent genetic alterations in HCC with non-linear models. Furthermore, we observe significant B cell enrichment in linear trees compared to non-linear trees suggesting the need for further research to uncover potential variations in immune cell types within genomically determined phylogeny types. These results hint at the possibility that tumor cells and their microenvironment may collectively influence the tumor evolution process.

Abstract 1964: Furmonertinib is a brain-penetrant EGFR TKI highly active in uncommon EGFR mutations, including PACC and exon 20 insertions

Abstract Background: Furmonertinib (AST2818, furmo) is an oral, highly brain-penetrant, and broadly active mutation-selective EGFR inhibitor against both classical and uncommon EGFR mutations (mts). Furmo has demonstrated promising interim efficacy and safety in non-small cell lung cancer (NSCLC) patients (pts) with EGFR exon 20 insertion (ex20ins) mts (Han et al., 2023). Furmo is currently being studied in a global Phase 3 trial for 1st line pts with ex20ins in NSCLC (FURVENT; NCT05607550) and has received FDA Breakthrough Designation in this pt population. P-loop and αC-helix Compressing (PACC) mts represent another subset of uncommon EGFR mts (Robichaux et al., 2021) that are similar to ex20ins in narrowing the drug binding pocket; include e.g. G719X, S768I, E709X, L747X, V774M; and are found as single or compound EGFR mts. Treatment options for pts with PACC mts remain limited. In this study, we further characterized PACC mts as cancer drivers in NSCLC and evaluated the pre-clinical activity and binding mechanisms of furmo in PACC and ex20ins mts. Methods: AACR Project GENIE and TEMPUS databases were used for mutation analysis. In vitro potency was determined in Ba/F3 cell lines for 33 PACC and 16 ex20ins mts. In vivo efficacy was conducted in pt-derived xenografts (PDXs). Molecular modeling was performed using MOE software. Results: PACC prevalence was 2.4% vs. 1.6% for ex20ins and 14% for classical EGFR mts in all NSCLC, corresponding to 10.4% PACC and 7% ex20ins of all EGFR mts. Co-mutation analysis revealed that other oncogenes, such as p53, co-occurred alongside PACC to a similar rate as with ex20ins and classical EGFR mts which further supports the role of PACC mts as a similar cancer driver as other EGFR mts in NSCLC. Ba/F3 cell lines harboring PACC mts were highly sensitive to furmo which showed overall superior potency vs. osimertinib and similar activity to afatinib. When comparing the mutant/wild-type IC50 ratio, furmo showed promising and high activity against single (e.g. G719X) and compound (e.g. ex19del+L718V) PACC mts. IC50 values were similar for PACC as compared to ex20ins mts. Therefore, furmo is predicted to be broadly active across both PACC and ex20ins mutant pt populations at clinically achievable drug levels. Molecular modelling revealed that furmo’s interaction with ex20ins and PACC mts is stabilized through hydrogen bonds with specific sites and its proximity to hydrophobic residues including L792 and P794 in the drug binding pocket. To further characterize furmo sensitivity in vivo against PACC mts, studies in PDXs are ongoing to evaluate efficacy, pharmacokinetics and pharmacodynamics and will be presented. Conclusions: PACC mts are important and prevalent cancer drivers in NSCLC and furmo is similarly active preclinically against both PACC and ex20ins mts. The activity of furmo in EGFR PACC mutant NSCLC is currently being evaluated in a global study FURTHER (NCT05364073). Citation Format: Monique B. Nilsson, Zineb Mounir, Luna Musib, Junqin He, Xiaoxing Yu, Qing Li, Huibing Luo, John V. Heymach, Jerry Y. Hsu, Stuart Lutzker, Xiuning Le, Marcin Kowanetz. Furmonertinib is a brain-penetrant EGFR TKI highly active in uncommon EGFR mutations, including PACC and exon 20 insertions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1964.

Abstract 1252: AI-derived predictions improve identification of real-world cancer driver mutations

Abstract AI-based variant effect predictors (VEPs), such as AlphaMissense, which is based on the protein structure prediction method AlphaFold, have gained significant attention for their potential to predict pathogenic effects of mutations. However, the utility of these methods for identifying pathogenic mutations in cancer remains unknown. We sought to quantify the prevalence of variants of unknown significance (VUSs) across the pan-cancer, multi-institution AACR GENIE cohort (N=160,965 samples) and test the utility of VEPs (SIFT, PolyPhen, MutationAssessor, REVEL, CADD, and AlphaMissense), using publicly available mutation annotations from each method, for 1) Annotating known pathogenic somatic cancer variants across the GENIE cohort, using known benign single nucleotide polymorphisms as negative controls 2) Identifying VUSs associated with overall survival (OS) in two cohorts of patients with non-small cell cancer (NSCLC), the MSK-IMPACT NCSLC (N=8,690) and non-MSK AACR GENIE Biopharma Collaborative NSCLC (N=977) cohorts, and 3) Identifying VUSs associated with known genomic patterns of mutual exclusivity in the MSK-IMPACT NSCLC cohort. In the GENIE cohort, 79% of mutations identified were VUSs according to an FDA-recognized molecular knowledge database, with a wide range of VUS frequencies across genes. Among VEPs trained without prior human-annotated knowledge, CADD and AlphaMissense had the highest AUROCs (0.940, 95%CI 0.937-0.943 and 0.917, 95%CI 0.913-0.922 respectively) for predicting known cancer drivers, demonstrating the power of protein structure modeling or functional genomic data for enhancing cancer drivers prediction. All VEPs identified known driver mutations in tumor suppressor genes more accurately than oncogenes, highlighting the challenge in identifying gain-of-function mutations. We tested whether VUSs reclassified as pathogenic by VEPs are associated with prognosis using inverse probability of treatment-weighted Cox’s propensity hazard models controlling for tumor mutational burden, treatment history, and other factors. VUSs reclassified as pathogenic by AlphaMissense and other VEPs in KEAP1 and SMARCA4 correlated with worse OS, comparable to that of known oncogenic mutations in those genes. Patients with tumors containing VUSs annotated as benign had similar OS to patients without analogous driver mutations. Finally, VUSs reclassified as oncogenic by AlphaMissense in genes within the RTK/RAS and NRF2 pathways followed expected patterns of mutual exclusivity, further suggesting biological validity. Despite not being trained to predict somatic effects in cancer, AI-derived mutation annotations can broaden the subset of annotated pathogenic variants in cancer and contribute to a more complete understanding of cancer genetics. Real-world outcomes and alteration patterns are additional benchmarks to consider when assessing the utility of VEPs in cancer. Citation Format: Thinh N. Tran, Chris Fong, Karl Pichotta, Anisha Luthra, Ronglai Shen, Yuan Chen, Michele Waters, Susie Kim, Gregory Riely, Debyani Chakravarty, Nikolaus Schultz, Justin Jee. AI-derived predictions improve identification of real-world cancer driver mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1252.

Abstract 2440: Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq

Abstract Unmet needs in cancer risk assessment include the ability to predict rodent life-time tumor responses from short-term exposures and a scientific basis for rodent to human extrapolation. As an approach to address these needs, the use of hotspot cancer driver mutations (CDMs) as biomarkers of cancer risk was integrated with an error-corrected, next generation sequencing (NGS) approach. We have developed and investigated the sensitivity of a high-throughput NGS method capable of detecting a panel of the most prevalent point mutations observed in human oncogenes and tumor suppressor genes. This method, CarcSeq, involves performing multiple, high-fidelity PCR reactions to amplify the most prevalent mutations reported to occur in human tumors, tagging the amplicons with 9 bp unique identifier sequences at each end, constructing libraries using Illumina ChIP Seq kits, sequencing the libraries on an Illumina NextSeq500 platform and generating single-strand consensus sequences (SSCS), bioinformatically, as a means of error-correction. We validated CarcSeq mutant fraction measurements through observations related to known cancer driver tissue-specificities and mutation spectra for normal human breast and lung, ductal carcinoma, and lung adenocarcinoma samples. Performance of a reconstruction experiment established CarcSeq has a sensitivity of 104. The approach has now been adapted to analyze analogous conserved codons in rat and mouse. For each of the three species, the developed panel includes 13-15 amplicons encompassing known cancer driver genes (e.g., KRAS, PIK3CA, and TP53) encompasses the equivalent of 30 known human hotspot codons and has a combined target sequence length of ~1 kb. Amplicons were generated from DNA isolated from 16-week-old, untreated rodent tissue samples with known differences in spontaneous tumor incidence: female Wistar, Sprague Dawley, and F344 rat mammary samples and male and female B6C3F1 and CD-1 mouse lung samples. The rodent panel was optimized to generate ~100,000 SSCS/amplicon. CDMs were observed at conserved codons in young, untreated rodents, and results are consistent with what was expected based on strain differences in spontaneous tumorigenesis. Citation Format: Kelly L. Harris, Binsheng Gong, Barbara Parsons, Jennifer Faske, Joshua Xu. Cancer driver mutations as quantitative biomarkers of cancer risk interspecies analyses using CarcSeq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2440.

Abstract 2162: Framework and pilot testing for the recruitment of a U.S.-based prospective cohort study of 100,000 Black women

Abstract Background: As of 2020, 46.9 million people in the United States (14.2% of the total population) identify as Black or African American. Collectively, Black Americans have the highest cancer death rate and shortest survival of any racial or ethnic group in the U.S. The drivers of cancer and other health conditions among Black women are likely multi-level, complex, and include contributions from environmental, behavioral, community, and system-level factors. Inadequate representation in research has limited the understanding of drivers of disparate outcomes, which, in turn, has hindered our ability to identify successful, culturally appropriate strategies for intervention. Approach: The American Cancer Society is establishing a cohort of at least 100,000 Black women to better understand cancer risk and outcomes in this population. The cohort will include two recruitment arms: (1) an “at risk” group of 85,000 participants between the ages of 25-55 years old with no prior cancer history and (2) a “survivorship” group of 15,000 participants previously diagnosed with breast, colorectal, or endometrial cancers. Pilot enrollment of the “at risk” group launched in October 2023 and will focus on understanding the disproportionate burden of early-onset cancer among Black women. For the “survivorship” group, pilot enrollment will launch in late 2024. The cancers of focus account for 95% of the absolute cancer mortality rate difference for Black vs. White women and offer the greatest opportunity for learning how to reduce cancer mortality disparities. Enrollment will focus on Black women who reside in the District of Columbia or one of 20 U.S. states representing >90% of the U.S. Black population. Results: Approximately 200 women were successfully consented into the cohort between October-November 2023. Study activities are conducted through an online portal optimized for mobile use. Through the portal, participants confirmed eligibility by completing an electronic survey, and reviewing and signing the informed consent. Upon consent, participants will complete a baseline survey and be sent follow-up surveys twice per year for at least 30 years. These surveys will track exposures, health outcomes, and participants’ lived experiences. Conclusions: Pilot work in 2023 demonstrated feasibility to engage Black women into a prospective cohort study. Based on the early feasibility success, full launch of enrollment is planned for May 2024. Citation Format: Alpa V. Patel, Lauren R. Teras, Elizabeth Kirkland, Jeshondria McCrary, Holly Vins, Jeuneviette E. Bontemps-Jones, Eunice Larbi, Maddison L. Hall, Peter J. Briggs, Daniela M. Dudas, Cari Lichtman, Lauren E. McCullough. Framework and pilot testing for the recruitment of a U.S.-based prospective cohort study of 100,000 Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2162.

Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

Abstract Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3938.

Abstract 4884: Evaluating the utility of in silico variant annotation tools for cancer driver detection

Abstract In cancer genomics, precise variant annotation is crucial for clinical decisions, drug development, and research. The burgeoning genomic data offers an opportunity to use data-driven approaches to generate knowledge that supports clinical decisions. Particularly machine learning (ML) and Deep learning (DL), are becoming essential, as their application is fast, scalable, simple to implement, and generates reproducible results. The methods ranging from simple sequence-based alignment scoring to advanced algorithms like Logistic regression, Support vector machine, and Recurrent neural networks, have been employed by multiple variant annotation tools. This study compares in-silico methods available in Ensembl's Variant Effect Predictor (VEP) using a test dataset with COSMIC annotations. ML/DL success relies on robust training sets with comprehensive genomic variants features, including effects on transcription/translation, genomic context, annotation resources, in silico pathogenicity predictions, and population allele frequency. The training set, composed of known benign or pathogenic variants, serves as a reference for these algorithms to classify new and unseen variants. Our analysis reveals a limited concordance between the prediction algorithms. Despite comparable true/false positives/negatives, discrepancies persist in variant classification. Certain algorithms exhibit a propensity to over or under-call deleterious mutations. Some demonstrate a tendency to classify random variants in non-cancer genes as deleterious. Challenges include the absence of consensus on informative features, diverse training datasets, and restriction to well annotated proteins/transcripts. Balancing the sensitivity and false positives in detecting cancer drivers is crucial. Integrating individual prediction scores with ML algorithms enhances tool performance but comes with risk of error propagation, and limited accuracy. The study emphasises the need for context-specific variant classification tools, as many variants' impacts are cancer-type specific, and some may drive disease synergistically. Existing tools, designed for a "one variant - one score approach," struggle to capture complex associations, especially those dependent on changes in the tumour microenvironment. Highlighting areas for improvement, the study addresses the "black box" problem in decision processes. While limited interpretability might not hinder practical applications, tools should evolve to assess more complex associations guided by biology. Formal consensus, reference training datasets, and standards are deemed essential for developing next-generation tools. The envisioned context-dependent tools aim to streamline feature complexity, thereby mitigating the black box problem and advancing the accuracy and interpretability of cancer variant annotation. Citation Format: Madhumita, Zbyslaw Sondka, Jon Teague. Evaluating the utility of in silico variant annotation tools for cancer driver detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4884.

Abstract 3395: Polyisoprenylated cysteinyl amide inhibitors: Effects on RAS signaling pathway intermediates and viability of mutant KRAS African American lung cancer cell line

Abstract Mutated KRAS is the most common cancer driver gene in lung adenocarcinomas in all racial and ethnic groups, including Blacks/African Americans (B/AAs). Drugs developed to combat KRAS-driven cancers become less effective due to intrinsic changes in the cells resulting in resistance and relapse. Due to the enormous challenges encountered in developing direct KRAS inhibitors, agents targeting alternative downstream RAS mediator kinase enzymes of the MAPK and PI3K/AKT signaling pathways have been developed. However, other avenues that directly target KRAS and similar cancer drivers must be explored to obtain alternative effective therapies. PCAIs constitute a group of potential anticancer agents that we developed to specifically disrupt and suppress signaling by hyperactive G-proteins such as mutated KRAS. In previous studies, PCAIs stimulated MEK1/2, ERK1/2, p90RSK and AKT phosphorylation resulting in cell death. The present study aims to determine whether specific inhibitors of the kinases can reverse PCAIs-stimulated phosphorylation and cell death. PCAIs were thus tested against the KRAS mutant NCI- cell line H23 (from a B/AA patient) and compared to KRASG12C inhibitor Sotorasib and other kinase inhibitors drugs. The EC50 values for the PCAIs NSL-YHJ-2-27 and NSL-AB-45 were 4.1 ± 0.3 and 5.5 ± 0.1 μM, respectively, compared to Gefitinib (EGFR), Sotorasib (KRASG12C), Dabrafenib (BRAF), Selumetinib (MEK1/2), Ulixertinib (ERK1/2) and Ipatasertib (AKT) whose EC50 values were 83 ± 12.9 μM, 38 ± 5.5 μM, 33 ± 1.2 μM, 71 ± 5.3 μM, 11 ± 0.2 μM, 12 ± 1.0 μM, respectively. The results indicate that the PCAIs are more effective against NCI-H23 than the other kinase inhibitor drugs. Future work will determine the effects of co-treating cells with the PCAIs and the respective kinase inhibitors on cell viability and PCAIs-induced phosphorylation of the kinases. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI) and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI Citation Format: Justin K. Mensah-Mamfo, Kweku Ofosu-Asante, Jassy Mary Lazarte, Amarender Goud Burra, Sofia A. Lugo, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors: Effects on RAS signaling pathway intermediates and viability of mutant KRAS African American lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3395.

Abstract 5064: Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer

Abstract Introduction: Oncogenic KRAS mutations define subsets of non-small cell lung cancer (NSCLC) with unique clinicopathologic and genomic features, and the recent development of direct KRAS inhibitors has changed the treatment landscape of these patients (pts). However, whether KRAS amplification is oncogenic and defines unique subset of potentially targetable NSCLCs is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of NSCLCs which underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR-GENIE v.13, TCGA, and other 212 studies (Cohort 2). Whole transcriptome sequencing and quantitative proteomics data from the The Cancer Genome Atlas (TCGA) and the Cancer Cell Lines Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. KRAS doxycycline-inducible models were generated to determine in vitro and in vivo oncogenicity of wild-type KRAS overexpression. Results: Among 15,341 pts with NSCLC identified, KRAS amplification was identified in 355 (2.4%) cases. Compared to KRAS not amplified cases, pts with KRAS amplified NSCLC were more likely to be males (52.4% vs 45.6%, p=0.01), have a history of smoking (82.1% vs 64.2%, p<0.01), and higher median pack-years (35 vs 23.5, p<0.01). KRAS amplification was also associated with higher median aneuploidy levels (as fraction of genome altered) (30.4% vs 14.3%, p<0.01), tumor mutational burden (p<0.01), and increased median PD-L1 expression (20% vs 5%, p=0.01). KRAS amplified cases were enriched with KRAS, POLE2, SLC34A2 and PALB2 mutations, while EGFR mutations were mutually exclusive (Q<0.1). Among NSCLC samples with transcriptomic and proteomic profiling from the TCGA and CCLE cohorts, KRAS amplification was associated with significantly increased KRAS mRNA (p<0.01) and protein expression (p<0.01), compared to KRAS diploid samples. Among pts with clinical outcomes data available (N=9,335), median overall survival (OS) from the date of diagnosis was significantly shorter in pts with KRAS amplified vs KRAS diploid genotype in both KRAS mutant (HR: 0.71, P=0.02) and KRAS WT (HR: 0.74, P=0.02) NSCLCs. In KRAS doxycycline-inducible models (BEAS-2B cells) we demonstrated increased cell proliferation and MAPK activation upon doxycycline-induced KRAS expression. In vivo, sustained expression of KRAS in animals constantly fed with doxycycline-diet triggered tumor formation and development of metastasis, indicating that WT KRAS overexpression is oncogenic. Conclusion: KRAS amplification defines a novel subset of NSCLCs characterized by distinct clinicopathologic and genomic features, and worse survival. Developing novel therapeutic strategies to target KRAS amplification may improve outcomes in pts with NSCLC. Citation Format: Biagio Ricciuti, Sandra V. Michelina, Joao Alessi, Xinan Wang, Federica Pecci, Alessandro Di Federico, Malini Gandhi, Giuseppe Lamberti, Enrico Patrucco, Lynette Sholl, Andrew Aguirre, Chiara Ambrogio, Mark Magdi Awad. Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5064.

Abstract 1974: Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Various types of EGFR-activating mutations are one of the most important oncogenic drivers in non-small cell lung cancer (NSCLC). Among NSCLC patients with EGFR mutations, 5-10% of patients have EGFR exon 20 insertion (ex20ins) mutations that are insensitive to clinically available EGFR tyrosine kinase inhibitors (TKIs). Recently, as mobocertinib (EXKIVITY®) failed to improve progression-free survival in Phase 3 clinical trial, there remains a high unmet medical need to develop new drugs with wider therapeutic window to treat NSCLC harboring EGFR ex20ins mutations. Herein, we report a novel EGFR-TKI, TRX-211-399, specifically designed to target ex20ins mutations. TRX-211-399 potently inhibited the proliferation of not only Ba/F3 cells overexpressing near-loop (A767dupASV, S768dupSVD) and far-loop (H773insNPH, H773dupH) mutations but also patient-derived NSCLC cell line. In particular, TRX-211-399 demonstrated high selectivity against wild-type EGFR. With favorable DMPK profiles, once-daily oral administration of TRX-211-399 showed significant tumor regression in multiple Ba/F3 xenograft models carrying different EGFR exon20ins mutations. Furthermore, in a PC-9-luc cell intracranial xenograft model, TRX-211-399 induced substantial anti-tumor effects and prolonged survival without body weight loss. TRX-211-399 is currently undergoing further preclinical evaluation as a potential candidate for clinical development for patients with EGFR exon20ins mutations. Citation Format: Kwangwoo Chun, Sumin Lim, Joohyun Lee, Seulgi Choi, Seongrak Kim, Youngil Choi, Areum Park, Bohee Kim, Yongseo Park, Eunhyun Choi, Sungwon Lee, Koo Lee. Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1974.

Abstract 3040: Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer

Abstract Epithelial ovarian cancer (OC) is typically diagnosed at a late stage and, despite treatment with cytoreductive surgery and chemotherapy, 70-80% of patients relapse and eventually die due to the disease. Major challenges to improve patient outcomes are to overcome therapy resistance and to individually tailor therapy. Uncovering the molecular drivers of OC, especially in the most common subgroup high-grade serous (HGS), will help develop novel therapeutic strategies to meet these challenges. Using the Cancer Genome Atlas (TCGA) data, we observed that a locus in 3q26.2, near the EVI1 oncogene, was the most significantly amplified region in HGS OC, occurring in more than 80% of those tumors. We validated these findings in an OC dataset from the Hartwig Medical Foundation and in OC cell lines from the DepMap database. Furthermore, HGS OC had the highest mean copy number of the 3q26.2 locus co-occurring with high mean EVI1 expression among all tumor entities in the pan-cancer TCGA dataset. These EVI1 expression levels are significantly higher than in normal fallopian tube, from which HGS OC most likely arises. EVI1 overexpression promotes leukemogenesis in 3q26-rearranged acute myeloid leukemia (AML), as a result of chromosomal translocations that enable hijacking of hyperactive enhancers. Here, we hypothesize that EVI1 also plays a crucial role in OC development, but that its aberrant expression in this disease is driven by the amplification of yet undiscovered regulatory elements near EVI1. We showed that EVI1-positive OC cell lines, of which the majority are HGS, are EVI1-dependent and harbor a ~200 kb amplified region near EVI1. This region interacts with the EVI1 promoter (4C-Seq), is characterized by open chromatin (ATAC-Seq), and shows high levels of H3K27 acetylation (H3K27ac ChIP-Seq), suggesting the presence of an active enhancer. Furthermore, based upon H3K27ac signal, we identified within this region a super-enhancer of approximately 65 kb. In OC cell lines without detectable EVI1 levels or cell lines from other cancer subtypes, such as breast and colon cancer, this location had no active enhancer and did not loop to the EVI1 promoter. Altogether, these data point to the generation of a hyperactive super-enhancer driving EVI1 overexpression in OC. These findings emphasize the importance of epigenetic control in the regulation of EVI1 expression, as previously shown in 3q26-rearranged AML. Since OC tumor cell lines are strongly dependent on EVI1, interference with this super-enhancer, leading to loss of EVI1 expression, could be exploited to inhibit tumor growth. For this purpose, we generated GFP-tagged EVI1 cell line models (e.g. SKOV3), which will be used to discover small molecules to target the activity of this super-enhancer. In conclusion, we discovered a new mechanism of enhancer-mediated overexpression of the EVI1 oncogene as a molecular driver in ovarian cancer, with possible implications for therapy. Citation Format: Leonie Smeenk, Sabrina Kruse, Roger Mulet-Lazaro, Ingrid Boere, John Martens, Stefan Gröschel, Claudia Scholl, Stefan Fröhling, Ruud Delwel. Tumor-specific super-enhancer drives overexpression of EVI1 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3040.

Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6587.